Non-volatile signals and redox mechanisms are required for the responses of Arabidopsis roots to Pseudomonas oryzihabitans

Abstract Soil bacteria promote plant growth and protect against environmental stresses, but the mechanisms involved remain poorly characterized, particularly when there is no direct contact between the roots and bacteria. Here, we explored the effects of Pseudomonas oryzihabitans PGP01 on the root system architecture (RSA) in Arabidopsis thaliana seedlings. Significant increases in lateral root (LR) density were observed when seedlings were grown in the presence of P. oryzihabitans, as well as an increased abundance of transcripts associated with altered nutrient transport and phytohormone responses. However, no bacterial transcripts were detected on the root samples by RNAseq analysis, demonstrating that the bacteria do not colonize the roots. Separating the agar containing bacteria from the seedlings prevented the bacteria-induced changes in RSA. Bacteria-induced changes in RSA were absent from mutants defective in ethylene response factor (ERF109), glutathione synthesis (pad2-1, cad2-1, and rax1-1) and in strigolactone synthesis (max3-9 and max4-1) or signalling (max2-3). However, the P. oryzihabitans-induced changes in RSA were similar in the low ascorbate mutants (vtc2-1and vtc2-2) to the wild-type controls. Taken together, these results demonstrate the importance of non-volatile signals and redox mechanisms in the root architecture regulation that occurs following long-distance perception of P. oryzihabitans.


Introduction
Plants live in harmony with soil microbiome communities, with whom they are in constant chemical communication. Soil bacteria and fungi can influence plant growth and performance, particularly through effects exerted at the seedling stage (Zhang et al., 2022). Plant growth-promoting rhizobacteria (PGPR) are comprised of different orders of bacterial species. They not only modulate plant growth and root system architecture (RSA) but they also trigger host immune responses (Poitout et al., 2017;Shekhar et al., 2019). Soil-borne plant pathogens can be controlled by the status of the soil microbiome, in what is known as 'disease-suppressive soil effects', which rely heavily on competition for plant nutrients between the different microorganisms (Schlatter et al., 2017). PGPR also produce compounds such as cyclic lipopeptides, polyketides, and bacteriocins that can have a direct negative effect on soil pathogens (Andric et al., 2021).
PGPR modulate RSA by regulating the production of phytohormones such as gibberellic acid (GA), auxin [indole acetic acid (IAA)], abscisic acid, and salicylic acid (SA) (Yuhashi et al., 2000;Poitout et al., 2017;Niu et al., 2018). Some PGPR species such as Pseudomonas aeruginosa, Klebsiella spp., Rhizobium spp., and Mesorhizobium spp. secrete IAA and so directly regulate RSA (Ahemad and Kibret, 2014). Such mutualistic interactions enhance the capacity of roots to take up nutrients (Glick, 2012). PGPR also improve the solubilization of minerals such as phosphorus, zinc, and potassium, and increase iron sequestration by siderophore production. Several Rhizobium species secrete nitrogenases that improve the fixation of nitrogen in anaerobic soils, as well as releasing organic acids to increase phosphorus uptake (Yanni et al. 2001).
Reactive oxygen species (ROS) are important components of the phytohormone signalling pathways that control RSA (Manzano et al., 2014;Kong et al., 2018;Yamada et al., 2020;Eljebbawi et al., 2021). For example, the control of ROS accumulation is an important factor in the emergence of LR primordia and it also influences the number of pre-branch sites (Orman-Ligeza et al., 2016). Transcription factors such as ethylene response factor (ERF)109 (also called redox-responsive transcription factor 1) are crucial regulators of the responses of RSA to environmental cues through modulation of jasmonate (JA), ethylene, and ROS signalling (Cai et al., 2014;Matsuo et al., 2015). While the effects of PGPR on plant morphology have been extensively studied, little attention has as yet been paid to the roles of ROS and redox signalling in plant-bacteria interactions, particularly when there is no direct contact between the roots and bacteria.
The non-fermenting yellow-pigmented, Gram-negative, lactose-and oxidase-negative rod-shaped bacterium, Pseudomonas oryzihabitans PGP01 (also known as Chromobacterium typhiflavum and Flavimonas oryzihabitans), is an opportunistic human pathogen. This saprophytic bacterium has been isolated from a range of human wound and soft tissue infections, leading to septicaemia, prosthetic valve endocarditis, and peritonitis. It also lives freely in soils as well as on medical and other equipment (Keikha et al., 2019). In plants, P. oryzihabitans has been linked to panicle blight in rice (Hou et al., 2020) and to stem and leaf rot in muskmelon . However, other studies have shown that P. oryzihabitans PGP01 can exert a positive effect on root growth (Belimov et al., 2015;Cantabella et al., 2020). The aims of the present study were firstly to determine the effects of P. oryzihabitans on RSA in A. thaliana, secondly to characterize how perception of P. oryzihabitans alters the root transcriptome profile, and thirdly to determine whether ROSrelated mechanisms were involved in the responses of RSA to perception of the presence of the bacterium.

Inoculation of bacteria onto plates containing Arabidopsis seedlings
The growth-promoting bacterium P. oryzihabitans strain PGP01 was obtained from the IRTA Postharvest Plant Growth Promoter Microorganism (PGPM) Collection (Lleida, Catalonia, Spain). Bacteria were grown in nutrient yeast dextrose agar (NYDA: nutrient broth, 8 g l -1 ; yeast extract, 5 g l -1 ; dextrose, 10 g l -1 ; and agar, 20 g l -1 ) media for 48 h. Bacteria were applied to plates containing 6-day-old Arabidopsis seedlings according to the method of Zamioudis et al. (2013). Bacteria were collected in 10 mM MgSO 4 , and washed by centrifugation at 5000 g for 5 min. After resuspension in 10 mM MgSO 4 , the bacterial concentration was adjusted to 1 × 10 6 by measuring turbidity at 600 nm. Aliquots (50 µl) of bacteria were applied at a distance of 5 cm from the root tip of 6-day-old Arabidopsis Col-0 seedlings. A concentration of 1 × 10 6 colony-forming units (CFU) ml -1 was used to examine the effects of the presence of bacteria on root architecture.
For the experiments designed to determine whether volatile signals were involved in root responses to P. oryzihabitans, 1 cm sections of the agar were removed from plates so as to physically separate the agar containing seedlings from the agar containing bacteria, as illustrated in Supplementary Fig. S1.

Measurements of root architecture
After 7 d of co-culture with bacteria, pictures of control and bacteriatreated plates were taken, and different parameters such as primary root (PR) length, number of visible LRs, and length of LRs were measured using ImageJ software. LR density was calculated by dividing the number of LRs by the PR length for each root analysed, as described previously (Dubrovsky and Forde, 2012). The LR density method provides a measure of the number of LRs per unit length of PR and allows a comparison of LR formation in PRs with different elongation rates.

RNAseq analysis
The roots of Arabidopsis seedlings were harvested after 7 d growth in the absence or presence of bacteria and immediately frozen in liquid nitrogen. Each biological replicate contained roots from at least three plates, each of them with six seedlings. RNA was extracted from frozen root samples using TRIreagent® (SigmaAldrich). RNA quality was checked by Nanodrop, and RNA integrity was confirmed using a 0.8% agarose gel. RNAseq data were analysed as described previously (De Simone et al., 2017).

Statistical analysis
All of the experiments were repeated at least three times. Data represent the mean ±SE of the mean. Data from the experiments using Col-0 and bacteria were analysed by one-way ANOVA and also by a pairwise t-test. A two-way ANOVA was also performed on the data from studies on SL, ascorbate, and GSH mutants. Statistical significance was judged at the level P<0.05, and Duncan's post-hoc test was used for the means separation when the differences were significant using the IBM SPSS statistics 25 program.

Results
Previous studies have shown that the presence of P. oryzihabitans PGP01 induces modifications in Pyrus and Prunus rootstocks (Cantabella et al., 2020(Cantabella et al., , 2021. The data presented in Fig. 1 demonstrate that perception of P. oryzihabitans PGP01 also induces changes in RSA in Arabidopsis. In these studies, P. oryzihabitans was placed on the same plates but not touching the roots of the Arabidopsis seedlings ( Fig. 1). Transcriptome profile comparisons of the roots of seedlings grown on plates in the absence or presence of bacteria were measured 7 d after plating ( Fig. 2A; Supplementary Table S1). The RNAseq analysis revealed the absence of bacterial transcripts from the roots of Arabidopsis plants (Supplementary Table S1). In total, 409 transcripts were increased in abundance in the roots grown in the presence of P. oryzihabitans compared with those grown in the absence of bacteria, and 201 transcripts were less abundant (Fig. 2B).
Further analysis of the most enriched GO terms revealed that transcripts encoding some hormone-related proteins were more expressed in roots exposed to P. oryzihabitans (Fig. 5C). These include ERF2, ERF107, DORMANCY/AUXIN AS-SOCIATED FAMILY PROTEIN 2 (DRM2), and KISS ME DEADLY 4 (KMD4) (Fig. 5A). Several transcripts associated with hypoxia responses (Fig. 5B) and nutrient acquisition and transport (Fig. 5C) were also increased in roots exposed to P. oryzihabitans.

Root responses to bacteria in lines with modified expression of ERF109
To analyse the role of ERF109 in root responses to P. oryzihabitans, RSA was compared in WT Arabidopsis seedlings, a transformed line overexpressing ERF109 (ov32), and a mutant line lacking a functional transcription factor (erf109; Fig. 6). The presence of bacteria increased LR density only in the WT (Fig.  7). LR density was not changed by perception of the bacteria in the ov32 plants or the erf109 mutants (Fig. 7).

Root responses to bacteria in ascorbate-deficient mutants
Two independent lines of ascorbate-deficient, vitamin C (vtc2) mutants were used to analyse the role of this low molecular weight antioxidant buffer in root responses to P. oryzihabitans (Fig. 8). LR densities were similar in all genotypes in the absence of bacteria (Fig. 8B). Moreover, the presence of P. oryzihabitans significantly increased LR density in all genotypes (Fig. 8).

Root responses to bacteria in glutathione-deficient mutants
Three independent lines of GSH-deficient mutants [phytoalexin-deficient 2 (pad2-1), the cadmium-sensitive 2 (cad2-1), and the regulator of APX2-1 (rax1-1)], which accumulate less glutathione (~30%) than the WT (Schnaubelt et al., 2015) were used to analyse the role of the low molecular weight antioxidant in root responses to P. oryzihabitans. The PRs of all genotypes were not significantly changed by the presence of P. oryzihabitans (Fig. 9A). Moreover, the presence of P. oryzihabitans significantly increased LR density in the WT roots but not in those of the cad2-1, pad2-1, and rax1-1 mutants (Fig. 9B).

Root responses to bacteria in SL-deficient mutants
The presence of bacteria increased LR density only in the WT. LR density was not changed by perception of the bacteria in mutants defective in SL synthesis or SL signalling (Fig. 10B). LR density was decreased in the WT in the presence of the synthetic SL GR24 but increased in the presence of GR24 and bacteria (Fig. 10D). In contrast, LR density was not significantly increased in the presence of GR24 and bacteria in any of the SL mutant lines (Fig. 10D). Moreover, bacteria-induced decreases in LR density were observed in the presence of GR24 in the roots of the max 4-1 mutants (Fig. 10D).

Root system architecture responses to P. oryzihabitans do not appear to be triggered by volatile signals
To test whether volatile signals were involved in the interactions between P. oryzihabitans and Arabidopsis roots, 1 cm sections of the agar were removed from the plates. Thus, the agar containing seedlings was physically separated from the agar containing bacteria (Supplementary Fig. S1). PR lengths (Fig.  11A) and LR densities (Fig. 11B) were similar in seedlings separated by a 1 cm gap in the agar (Control), separated from seedlings grown in the presence of P. oryzihabitans (Plants and bacteria), or separated from agar on which P. oryzihabitans was grown (Plants/bacteria).

Discussion
RSA undergoes fine tuning in response to cues from the soil microbiome (Hodge et al., 2009;Ruiz Herrera et al., 2015). For example, the presence of PGPR modifies RSA and primes plant defences against pathogens and herbivores through induced systemic resistance responses (Pieterse et al., 2014;Rashid et al., 2017;Veselova et al., 2019). The data presented here demonstrate that remodelling of the root transcriptome and RSA occurs upon perception of P. oryzihabitans, without direct contact between the bacteria and the roots. However, root cap-derived signals from the soil microbiome were found to be important in the regulation of RSA (Crombez et al., 2020). The root responses to P. oryzihabitans reported here involve subtle transcriptome remodelling and require SLs and redox signalling through GSH and ERF109, but not ascorbate. The RSA response was lost once the agar containing the seedlings was physically separated from that containing the bacteria, suggesting that volatile signals are not important drivers of root remodelling. Considerable genetic variation in the ability of Arabidopsis accessions to benefit from root associations with P. simiae has been reported (Wintermans et al., 2016). Pseudomonas species deploy a range of signals that modulate root development, including the secretion of phytohormones such as IAA and other small molecules, and the release of volatile organic compounds (VOCs; Zamioudis et al., 2013). For example, P. fluorescens SS101 promotes plant growth through the release of 13-tetradecadien-1-ol, 2-butanone, and 2-methyl-n-1-tridecene (Park et al., 2015) while P. putida and P. fluorescens produce cyclodipeptides such as cyclo(l-Pro-l-Val), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Tyr), which modulate the expression of auxin-responsive genes in roots (Ortiz-Castro et al., 2020). Pseudomonas oryzihabitans PGP01 is able to produce IAA, when supplied with appropriate substrates (Cantabella et al., 2021). Like other Pseudomonas strains, P. oryzihabitans PGP01 triggers auxin-dependent root developmental programmes including abundant LR formation (Ortiz-Castro et al., 2011Zamioudis et al., 2013). The data presented here suggest that non-volatile signals are essential for the control root responses to P. oryzihabitans PGP01.

2015)
, which is also highly expressed in roots exposed to P. oryzihabitans. Ethylene promotes the homeostasis of Na + /K + , nutrients, and ROS to enhance plant tolerance to salinity (Tao et al., 2015).
The perception of P. oryzihabitans caused changes to the root transcriptome even though there was no direct colonization or physical contact between the organisms except through the agar. The genes that were most highly expressed in response to P. oryzihabitans include mRNAs encoding GDSL28 and HSFA6b. HSFA6b plays a pivotal role in pant responses to abscisic acid and in thermotolerance (Huang et al., 2016) as well as ROS accumulation and the expression of antioxidant genes (Wenjing et al., 2020). Other transcripts that were increased in abundance include DRM2, which is important in plant Fig. 6. Representative images of wild-type Arabidopsis seedlings, seedlings overexpressing ERF109 (ov32), and erf109 mutants. Seedlings had been grown for 6 d in the absence of P. oryzihabitans and then for a further 7 d in either the absence (control) or the presence of bacteria (PGP01). Col-0 ov32 erf109 *** Fig. 7. The effect of the presence of P. oryzihabitans PGP01 on primary root length (A) and lateral root density (B) in wild-type A. thaliana, a transgenic line overexpressing redox-responsive transcription factor 1 (ov32), and a erf109 mutant line. Samples of bacterial inoculum was placed 5 cm away for the tips of the primary roots of 6-day-old seedlings that had been grown on agar plates. Root parameters were measured 7 d after inoculation. Data show the mean ±SE of three independent biological samples. Asterisks indicate significant differences according to t-test (P<0.05).
defence responses (Roy et al., 2020), and KMD4, which targets type-B ARR proteins for degradation and is required for cytokinin responses through control of transcription factors (Kim et al., 2013). Transcripts encoding enzymes and proteins involved in plant responses to hypoxia, such as unknown proteins 26 and 32, were increased in roots exposed to P. oryzihabitans (Fig. 5B). Severe oxygen depletion can suppress LR formation (Shukla et al., 2019;Pedersen et al., 2021). The uptake of oxygen in respiration by the bacteria may contribute to some of the observed metabolic adaptations in the transcriptome signature (Pucciariello and Perata, 2021). Other genes that were highly expressed in the presence of bacteria encode proteins that are involved in nutrient acquisition and transport. For example, the levels of transcripts encoding several root hair-specific proteins including RHS7, RH17, and RH18, and a number of transporters such as the sucrose transporter SUC2, the POLYOL/ monosaccharide transporter PMT6, the boron transporter 1 BOR1, the aluminium-activated malate transporter ALMT1, and the zinc transporter 3 precursor ZIP3 were higher in roots in the presence of P. oryzihabitans. Similarly, the levels of transcripts encoding HAK5 that is required for plant growth and K + acquisition particularly under saline conditions (Nieves-Cordones et al., 2010) were significantly higher in the roots exposed to P. oryzihabitans, as were transcripts encoding the MYB transcription factor MYBL2, which is a key negative regulator of anthocyanin biosynthesis in response to changes in sucrose availability (Dubos et al., 2008).
The expression of genes encoding UDP-glycosyltransferases UGT91A1, UGT78D4, UGT84A1, and UGT78D1, as well as those encoding transparent testa TT7 and GST26, which play an important role in regulating the availability of secondary metabolites, was lower in bacteria-exposed roots. Similarly, transcripts encoding GA3OX, which catalyses the conversion of precursor GAs to their bioactive forms during vegetative growth (Mitchum et al. 2006), were significantly lower in the roots exposed to P. oryzihabitans.
Targeted ROS production is crucial to the hormone-dependent regulation of RSA (Eljebbawi et al., 2021). For example, hyrdogen peroxide is required for brassinosteroid-mediated cell division in the root quiescent centre and for seedling development . The data presented here provide evidence that ROS signalling is important in RSA responses to P. oryzihabitans. For example, while levels of ERF109  1 and vtc2-2). Samples of bacterial inoculum were placed 5 cm away for the tips of the primary roots of 6-day-old seedlings that had been grown on agar plates. Root parameters were measured 7 d after inoculation. Data show the mean ±SE of three independent biological samples. Asterisks indicate significant differences according to t-test (P<0.05). Col-0 pad2-1 cad2-1 rax1-1 Col-0 pad2-1 cad2-1 rax1-1 * Fig. 9. The effect of the presence of P. oryzihabitans PGP01 on primary root length (A) and lateral root density (B) in wild-type A. thaliana and mutants that are defective in glutathione (cad2-1, pad2-1, and rax1-1). Samples of bacterial inoculum were placed 5 cm away for the tips of the primary roots of 6-day-old seedlings that had been grown on agar plates. Root parameters were measured 7 d after inoculation. Data show the mean ±SE of three independent biological samples. Asterisks indicate significant differences according to t-test (P<0.05).
transcripts were not changed in the roots exposed to bacteria, the P. oryzihabitans-induced changes in RSA were absent from the erf109 mutants. ERF109 is involved in the amplification of ROS signalling and systemic transmission of ROS signals in response to biotic and abiotic stresses (Bahieldin et al., 2016), as well as in the JA-dependent regulation of RSA (Xu et al., 2020).
The P. oryzihabitans-induced changes in RSA were similar in the vtc mutants that are deficient in the low molecular weight antioxidant ascorbate (Foyer et al. 2020) and the WT plants. This finding demonstrates that changes in total antioxidant capacity alone are not important in plant-bacteria interaction. The vtc mutants have modified phytohormone signalling pathways (Kerchev et al., 2013;Caviglia et al., 2018) but these changes do not influence the responses of RSA to P. oryzihabitans. In contrast, the P. oryzihabitans-induced changes in LR density were absent from the cad2-1, pad2-1, and rax1-1 mutants, indicating that GSH-mediated redox regulation is important in root responses to the bacterium. GSH is essential for root development (Passaia et al., 2014, Ehrary et al., 2020. The GSH-deficient rootmeristemless1 (rml1) mutant is unable to develop roots because of impaired root apical meristem functions (Vernoux et al., 2000). The glutathione reductase-deficient miao mutants also show poor root growth  Fig. 10. The effect of the presence of P. oryzihabitans PGP01 on primary root length (A) and lateral root density (B) in wild-type A. thaliana and mutants that are defective in SL synthesis (max3-9 and max4-1) and signalling (max2-3). Samples of bacterial inoculum were placed 5 cm away for the tips of the primary roots of 6-day-old seedlings that had been grown on agar plates. Root parameters were measured 7 d after inoculation. Data show the mean ±SE of three independent biological samples. Asterisks indicate significant differences according to t-test (P<0.05).  Fig. 11. The effect of removal of the agar between P. oryzihabitans PGP01 and Arabidopsis seedings. Arabidopsis seedlings were either separated by a 1 cm gap in the agar (Control), separated by a 1 cm gap from seedlings grown in the presence of P. oryzihabitans (Plants and PGP01), or separated from agar on which P. oryzihabitans was grown (Plants/PGP01). Seedlings were grown for 6 d in the absence of P. oryzihabitans and then for a further 7 d in either the absence or presence of bacteria. Primary root length (A) and lateral root density (B). (Yu et al., 2013). Mutants lacking glutathione peroxidases have modified root phenotypes (Passaia et al., 2014). Crucially, glutaredoxins (GRXs) such as GRXS8 and GRXS17 are involved in the regulation of RSA (Ehrary et al., 2020;Martins et al., 2020). GSH enhances the sensitivity of roots to auxin (Pasternak et al., 2020) and is required for the conversion of indole butyric acid (IBA) to IAA (Trujillo-Hernandez et al., 2020). The data presented here demonstrate that the root GSH pool is essential for the facilitation of bacteriadriven changes in RSA. The GSH pool is involved in the SL-dependent control of RSA through the MAX2 protein (Marquez-Garcia et al., 2014). SLs are important in rhizosphere communication (Bouwmeester et al., 2007) and are required for plant responses to nutrient deficiencies (Shindo et al., 2020). They are required for the initiation of symbiotic interactions with arbuscular mycorrhizal fungi, when nutrients are limiting (Akiyama et al., 2005;Aliche et al., 2020). The bacteria-induced increases in LR density were absent from mutants that are defective in SL synthesis or signalling, demonstrating the essential role of these phytohormones in plant-bacteria interactions.
In summary, evidence is presented showing that the root system of A. thaliana seedlings is changed in the presence of P. oryzihabitans PGP01 in a manner that suggests that this bacterium functions as a PGPR. Moreover, the observed changes in the root transcript profile are due to increases in mRNAs encoding proteins involved in mineral nutrition and phytohormone signalling but not defence or immune responses. Crucially, the data show that the long-distance perception of P. oryzihabitans PGP01 is sufficient to modulate RSA. ERF109, SLs, and GSH are key components required for the bacteriamediated control of RSA. These findings demonstrate that SL and redox signalling are important factors in root responses to P. oryzihabitans, but changes in antioxidant capacity alone do not influence this process.

Supplementary data
The following supplementary data are available at JXB online. Fig. S1. Representative images of wild-type Arabidopsis seedlings that were separated by a 1 cm gap in the agar, by a 1 cm gap from seedlings growing in the presence of P. oryzihabitans, or separated from agar on which P. oryzihabitans was grown. Table S1. Bacteria-induced changes in differentially expressed genes in Arabidopsis thaliana roots.